Biomolecular condensates can enhance pathological RNA clustering

Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which drives a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.


Introduction
Macromolecular phase separation is a segregative mechanism utilized by living cells to form mesoscale compartments, henceforth referred to as biomolecular condensates 1,2,3,4,5,6 .A common feature of many ribonucleoprotein (RNP) condensates, such as the cytoplasmic stress granules, is their dynamic uid-like material properties, which enable their facile on-demand formation, dissolution, and rapid macromolecular transport 7 .Experimental and computational approaches probing intra-condensate rheology have revealed that biomolecular condensates are viscoelastic uids 8,9,10,11 with material and structural properties that can change over time, termed physical aging 12 .Age-dependent loss in uidity of condensates can lead to a liquid-to-solid phase transition 12,13,14,15,16 .Both protein and RNA components can contribute to the physical aging of condensates.In the protein-centric model of condensate aging 17,18,19,20 , phase separation of RNA-binding proteins (RBPs) such as hnRNPA1, FUS, and TDP43 is considered metastable 9,12,14,21,22,23,24 .Hence, condensates formed by these RBPs are susceptible to transition into stable solids, which can either be a glassy material 9 , viscoelastic Kelvin-Voigt solid 12 , or amyloid ber 23,25 .Clinically relevant mutations in these RBPs linked to numerous neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) 13,15,22,24 can accelerate the physical aging of condensates.
Multiple lines of recent evidence also suggest that RNA-driven condensation plays a central role in the formation and regulation of RNP condensates including stress granules 26,27,28 and paraspeckles 29,30 .Furthermore, aberrant intracellular aggregation of GC-rich RNAs with expanded repeats is a hallmark of numerous neurological disorders including Huntington's disease and ALS 31,32,33,34,35,36,37 .These RNAs form pathological intracellular condensates, which are sensitively dependent on the repeat length and likely involve sequence and structure-speci c homotypic RNA-RNA interactions 38,39,40 .Homotypic RNA clustering has also been reported to drive mRNA self-assembly in germ granules 41,42 and in vitro for RNA homopolymers 26 , indicating condensation of RNA molecules may be ubiquitous in cells.A recent study showed that RNAs have an intrinsic propensity to undergo protein-free phase separation in vitro upon heating with lower critical solution temperatures (LCSTs), driven by desolvation entropy and Mg 2+dependent physical cross-linking of RNA molecules 43 .The thermo-responsive phase separation of RNAs can be coupled to an intra-condensate networking transition within the dense phase, referred to as percolation 44,45,46 , which engenders long-lived physical cross-linking of RNA chains through nucleobasespeci c interactions.Importantly, phase separation and percolation of RNA chains are two distinct transitions, the former being an entropy-driven density transition leading to the formation of phaseseparated RNA condensates and the latter being an associative transition mediated by multivalent RNA-RNA interactions.Percolation coupled to phase separation can result in the dynamical arrest of RNAs in the dense phase rendering RNA condensation irreversible.RNA percolation could potentially be exploited in disease conditions to perturb RNP granule dynamics via aberrant RNA clustering, and hence, provides a conceptual framework to model the aggregation landscape of repeat expanded RNAs.These outstanding insights lead to two important questions: (a) do multicomponent RNP condensate microenvironments enhance or suppress RNA percolation, and (b) does intra-condensate RNA percolation contribute to the age-dependent condensate transition from a predominantly liquid to a solid state?
To address these questions, here we employ a designed multi-component condensate system and quantitative microscopy with nanorheology, focusing our attempt to capture key elements of intracondensate RNA percolation.Our experiments reveal that percolation transitions of physiologically relevant and disease-associated repeat RNAs engender the formation of viscoelastic RNA-rich subphases embedded within a uid-like condensate matrix in an age-dependent manner.The timescale of RNA percolation is tuned by the RNA sequence, secondary structure, and repeat length.Importantly, multivalent RBPs such as G3BP1 can buffer intra-condensate RNA-RNA homotypic interactions, thereby enhancing condensate metastability and delaying the onset of RNA clustering.Overall, our ndings suggest that biomolecular condensates can act as sites for RNA clustering and highlight the chaperonelike function of RBPs to buffer the intrinsic capacity of some RNAs to undergo irreversible percolation.

RNA aggregation is enhanced in multi-component biomolecular condensates
To study the emergent role of homotypic RNA-RNA interactions within heterotypic protein-nucleic acid condensates, we utilized a model condensate system amenable to quantitative experimental interrogation.This model system consists of an RNA binding motif-inspired multivalent disordered polypeptide [RGG; sequence: (RGRGG) 5 ] and a single-stranded nucleic acid [poly-thymine DNA, d(T) 40 ].
Phase separation in RGG-d(T) 40 mixtures is driven by obligate cation-π and electrostatic protein-nucleic acid interactions 8 .Furthermore, the material and physical properties of RGG-nucleic acid condensates are fully tunable via peptide and nucleic acid sequence and length 8,47 , providing a robust means to tune the condensate microenvironment.Introducing additional components, such as client RNAs featuring speci c primary sequence and secondary structures, to this model condensate allows us to systematically probe the effects of compositional complexity and RNA-driven changes on condensate physical properties (Fig. 1a).We hypothesize that the introduction of RNAs capable of forming homotypic intra-and inter-molecular contacts within RGG-d(T) 40 condensates would result in one of two outcomes: the formation of a homogenous ternary condensate where the RNA fully mixes with the two primary condensate-forming components, RGG and d(T) 40 , or a multiphasic condensate with RNA preferentially partitioning into one of the two co-existing phases.A possible third outcome would be a homogenous ternary condensate undergoing age-dependent transformation into a multiphasic condensate due to RNA demixing (Fig. 1a).In our rst set of experiments, we put this idea to the test by utilizing the naturally occurring telomeric repeat-containing RNA 48,49 [TERRA, sequence: (UUAGGG) n ] (Fig. 1b), which is known to form G-quadruplex (GQ) structure 50,51,52 .
Without multivalent cofactors such as Mg 2+ ions, (TERRA) 10 alone remains fully soluble over a period of 24 hours at room temperature (Fig. 1c).When introduced as a client to RGG-d(T) 40 condensates, (TERRA) 10 preferentially partitions in the dense phase and remains homogeneously distributed at initial time points (Supplementary Fig. 1).Using a nucleic acid-binding dye (SYTO-13), we next tracked whether intra-condensate spatial distribution of TERRA changes with condensate age.Remarkably, we nd that TERRA undergoes age-dependent demixing inside RGG-d(T) 40 condensates leading to the formation of RNA clusters (Fig. 1d).As a result, a distinct multiphasic architecture emerges that is de ned by an RNArich inner core surrounded by an RNA-deplete shell.Utilizing uorescently labeled anti-sense TERRA, we con rmed that the inner SYTO-13-positive sub-phases in aged condensates are indeed formed by TERRA clusters (Supplementary Fig. 1).The outer phase of aged condensates displays liquid-like dynamics such as fusion with the outer phase of nearby condensates, which is not observed for the RNA-rich core (Fig. 1d; Supplementary Video 1).To elucidate the molecular compositions of these distinct phases, we employ a pairwise imaging approach and identify the localization of each component.The TERRAenriched clusters within the condensate are signi cantly depleted of RGG (probed by Alexa594-labeled RGG) and d(T) 40 [probed by Cy5-labeled d(T) 40 ] (Fig. 1e-h).Line pro les illustrate the anti-correlation between TERRA and either of these two components (Fig. 1f, h).Moreover, the RNA clusters are shown to be quite irregular in morphology (Fig. 1i).Replacing d(T) 40 with r(U) 40 as a condensate forming component in the ternary system did not alter the age-dependent clustering of TERRA (Supplementary Fig. 2).However, we do not observe this time-dependent appearance of RNA clusters in the dense phase of binary condensates composed of TERRA and RGG (Supplementary Fig. 3).These observations suggest that age-dependent RNA demixing is an emergent property of the ternary condensates.Overall, these results demonstrate that RNA partitioning into multi-component biomolecular condensates can drive age-dependent homotypic RNA clustering (Fig. 1j).Next, we attempt to dissect which features of the RNA molecules enable this emergent behavior.
Intra-condensate RNA aggregation is driven by RNA percolation transition In the absence of proteins, RNA can undergo reversible temperature-dependent phase separation with a secondary percolation transition 43 (Fig. 2a).The percolation transition, which is manifested by homotypic RNA-RNA interactions and sensitively depends on the RNA sequence and secondary structure 43 , dynamically arrests RNA in the dense phase and makes RNA phase separation irreversible.
Therefore, RNAs with a strong percolation propensity tend to form irreversible condensates in contrast to RNAs with a weak or no percolation propensity (Fig. 2a).We hypothesized that the demixing of (TERRA) 10 in the dense phase of the multicomponent protein-nucleic acid condensates (Fig. 1; Supplementary Fig. 2) stems from a strong percolation propensity of the RNA.To test this idea, we performed temperature-controlled microscopy of (TERRA) 10 .We observed that (TERRA) 10 [1.0 mg/ml RNA in 50 mM HEPES (pH 7.5), 6.25 mM Mg + 2 ] remains homogenous at 20°C but undergoes an irreversible phase transition with a lower cloud point temperature (LCPT) of T = 60.2 ± 1.3°C upon heating.The irreversibility of the (TERRA) 10 condensates upon cooling to 20°C suggests the formation of a strongly percolated RNA network in the dense phase (Fig. 2b, c; Supplementary Video 2).Using a series of Mg 2+ titrations, we mapped the state diagram of (TERRA) 10 and observed that the phase separation coupled to percolation behavior of (TERRA) 10 is extremely sensitive to Mg 2+ concentration with a very narrow range of Mg 2+ concentrations (5.75 to 6.5 mM) where thermo-responsive phase separation is experimentally observable (Fig. 2c).At Mg 2+ concentrations higher than 6.5 mM, we found that (TERRA) 10 always exists as percolated clusters even at the lowest temperature tested, 2.0°C (Fig. 2c).At temperatures above percolation temperature (T prc ), (TERRA) 10 clusters underwent shape relaxation into energetically favorable spherical condensates that persist after cooling, thereby irreversibly trapping the RNA in the condensed state (Fig. 2d; Supplementary Video 3).These results therefore reveal a strong percolation propensity of (TERRA) 10 , which is likely to drive its age-dependent aggregation within RGGd(T) 40 condensates.
An important molecular feature of RNA percolation is the contribution of purine-mediated base-pairing and stacking interactions 43 .It is also known that G-rich sequences of RNA can form aggregates in solution 26 .Many of these sequences are also known to form GQ structures with high stability in the presence of monovalent ions 53 .We reasoned that the propensity of TERRA to form GQ structures 50,52 may contribute to homotypic RNA clustering via percolation.If true, this can be modulated by TERRA sequence perturbations.To test this idea, we employed a mutant (TERRA) 10 sequence [(mut-TERRA) 10 ] with a G-to-U substitution e.g., (UUAGUG) 10 , which is expected to disrupt the stability of the GQ state 54,55 .We performed temperature-controlled microscopy experiments with (mut-TERRA) 10 samples and observed that the LCST transition of (mut-TERRA) 10 requires a substantially higher concentration of Mg 2+ ions (Fig. 2b, c vs. 2e, f).At 25 mM Mg 2+ , (mut-TERRA) 10 undergoes reversible phase separation upon heating with an LCPT of 28.8 ± 0.42°C (Fig. 2e; Supplementary Video 4).We note that under the same condition, wild-type (WT) (TERRA) 10 would exist as percolated clusters at all experimentally accessible temperatures.Percolation of (mut-TERRA) 10 was not observed at any conditions tested (Fig. 2f) demonstrating that the G-to-U substitution attenuates the intermolecular RNA-RNA interactions between TERRA chains.Consistent with the absence of RNA percolation in RNA-only condensates, we nd that (mut-TERRA) 10 does not form age-dependent RNA aggregates in RGG-d(T) 40 condensates (Fig. 2g; Supplementary Fig. 4, 5).This data suggests that intra-condensate RNA percolation is RNA sequence-and structure-speci c.
Does the intra-condensate aggregation of TERRA, as reported in Fig. 1d-i, proceed via a transition from intra-molecular to inter-molecular GQ structures?We tested this idea by utilizing a GQ-selective uorescent probe Thio avin T (ThT), which has been previously shown to display uorescence activation upon binding to GQ structures with high speci city 56,57,58 .At an early time-point after the preparation of RGG-d(T) 40 condensates containing (TERRA) 10 , ThT uorescence was observed to be uniformly distributed throughout the condensate suggesting that TERRA is likely forming intramolecular GQs within the homogenous condensate (Fig. 2h).Importantly, RGG-d(T) 40 condensates with (TERRA) 10 clusters, formed upon aging, displayed ThT positivity, with uorescence intensities of the clusters being ~ 4-fold higher than nascent homogeneous condensates.This observation indicates that the TERRA in these aggregates is also likely to form GQs (Fig. 2h).In the case of the (mut-TERRA) 10 , there was no detectable ThT signal under identical imaging conditions at all time points (Fig. 2h).This is consistent with the previous reports that this particular G-to-U substitution disrupts the GQ structure of TERRA 54,55 .
Negative control of RGG-d(T) 40 and RGG-d(T) 40 condensates containing a non-percolating RNA 43 , polyuridylic acid (polyU), did not show any measurable ThT signal under identical experimental conditions (Supplementary Fig. 6).Finally, intra-condensate (TERRA) 10 clusters were observed ubiquitously in the presence of a wide variety of monovalent salts (Supplementary Fig. 7) that differentially impact the stability of RNA GQ structures, suggesting observed RNA percolation may directly stem from the clustering propensity of purine-rich RNAs 43 .

Timescale of RNA clustering depends on the TERRA repeat number
The number of guanine tracts (G-tracts) plays important roles in monomeric RNA GQ and multimeric RNA structure formation 59,60,61 .We reasoned that time-dependent TERRA aggregation in RGG-d(T) 40 condensates (Fig. 1d) can be tuned by the ability of TERRA to form multi-molecular GQ structures, which is expected to depend on the number of repeat units of TERRA 59,62 .To test this idea, we titrated the number of TERRA repeat units, [UUAGGG] n , where n = 1, 4, 6, and 10; far below the number of repeat units transcribed in cells, 100 to 9000 nucleotides 49,63 .We observed that all RNAs that could form intramolecular GQs, e.g., n = 4, 6, and 10, showed age-dependent intra-condensate cluster formation with a corresponding timescale correlated inversely with the number of repeats (Fig. 3a).(TERRA) 10 forms microscale aggregates the fastest with a timescale of ~ 2 hours, whereas (TERRA) 4 form clusters at substantially slower rate (~ 8 hours) and the clusters are less distinct (Fig. 3a).To quantify the relative intra-condensate cluster sizes as a function of time, we employed spatial autocorrelation analysis (SAC) of the confocal uorescence images of condensates (Fig. 3a insets, white-dotted boxes; Supplementary Fig. 8).In the absence of clusters larger than the detection limit of the microscope, which is the case for (TERRA) 6 at 15 minutes after sample preparation, SAC returns the size of spatial uctuations, 0.28 ± 0.01 µm, which closely corresponds to the image resolution (1 pixel = 0.2196 µm).However, at 18 hours time point when the RNA clusters in the condensate are detectable, SAC reveals the characteristic size of the RNA clusters being 0.89 ± 0.04 µm (Fig. 3b).Employing SAC, we nd that the size of RNA clusters increases, and the timescale of RNA cluster formation decreases with increasing number of repeat units of TERRA (Fig. 3c, d).Simultaneously, we see increased negative correlation between the intensities of TERRA and the primary components, RGG and d(T) 40 , as a function of condensate age (Fig. 3e, f; Supplementary Fig. 9, 10).As clusters become more pronounced with the aging of condensates, the anticorrelation of intensities becomes more apparent (Fig. 3e, f; Supplementary Fig. 9, 10).These data suggest that percolated TERRA clusters demix from the RGG and d(T) 40 in a time-dependent manner.
Together with the data presented in the previous section on (mut-TERRA) 10 failing to form homotypic intra-condensate clusters, these data suggest that repeat number and RNA sequence variations are molecular regulators of age-dependent RNA percolation within biomolecular condensates.
Repeat expanded RNAs form intra-condensate clusters in a length-dependent manner Intracellular RNA aggregation has been widely implicated in several diseases primarily in the category of trinucleotide repeat expansion disorders such as Huntington's disease (CAG repeat), Fragile-X syndrome (CGG repeat), and myotonic dystrophy (CUG repeat) 37,64,65,66,67,68,69 .One characteristic of these disorders is that pathology occurs at a number of repeats beyond what is found in healthy individuals.Further, the threshold number of repeats associated with pathological outcomes varies with the repeat RNA sequence.For example, the number of r(CAG) repeats required for disease onset is ~ 15 to 20 repeats less than that required for r(CUG) (Fig. 4a).This implies that the cellular machinery can better tolerate r(CUG) repeats than it can with r(CAG) repeats 64, 65, 66, 67, 68 .
Motivated by our results of enhanced TERRA clustering as a function of increasing repeat number, we interrogated the clustering propensity of two repeat RNAs, r(CAG) n and r(CUG) n , with repeat lengths higher and lower than the pathological limit using our model condensate system.r(CAG) repeat lengths lower than 21 are found in healthy individuals, however, repeat lengths exceeding this number are considered to be at the intermediate-high risk scale of being most likely to attain diseases such as spinocerebellar ataxia and Huntington's disease (Fig. 4a).We observed that r(CAG) 20 stays predominantly homogeneous in the dense phase of RGG-d(T) 40 condensates at all ages till 24 hours, the end time of our experiments.The mean RNA cluster size is estimated to be 0.28 ± 0.01 µm, which closely corresponds to the image resolution (1 pixel = 0.2196 µm) (Fig. 4b, c; Supplementary Fig. 11).Strikingly, r(CAG) 31 , which corresponds to a pathological number of repeats, spontaneously demixes into fractallike clusters prior to sample imaging (15 minutes post-preparation) (Fig. 4b, c; Supplementary Fig. 11).In these samples, r(CAG) 31 clusters are extensively percolated with cluster sizes that are nearly 4-fold larger (3.94 ± 0.14 µm) than those formed by TERRA at the same concentration (Fig. 4c).Reduced r(CAG) 31 concentration reduces the intra-condensate cluster size, but the RNA aggregation timescale appears to be independent of r(CAG) 31 concentration (Supplementary Fig. 12).
We next examined intra-condensate percolation of r(CUG) n for n = 31, which represents a healthy number of repeats.Similar to r(CAG) 20 , we did not observe intra-condensate r(CUG) 31 clustering.The estimated cluster size is 0.40 ± 0.02 µm, which closely corresponds to the image resolution (1 pixel = 0.2196 µm) (Fig. 4d, e; Supplementary Fig. 11).In stark contrast, r(CUG) 47 forms microscale clusters within 15 minutes after sample preparation (Fig. 4d, e; Supplementary Fig. 11).We also tested intra-condensate clustering behavior of a hexanucleotide repeat expanded RNA, r(GGGGCC) n , that is associated with ALS and frontotemporal dementia (FTD) 59,70,71 .We observed spontaneous r(GGGGCC) 5 clustering in RGGd(T) 40 within 15 minutes after sample preparation (Supplementary Fig. 13).Importantly, r(GGGGCC) 5 clusters showed ThT positivity but not r(CAG) 20 or r(CAG) 31 clusters (Supplementary Fig. 14).This is consistent with the ability of r(GGGGCC) 5 to form GQ structures 59 .Overall, the distinct differences in the intra-condensate percolation behavior of each of these RNAs and TERRA of various lengths suggest a heuristic framework where a sequence and length-speci c activation energy barrier dictates the timescale of intra-condensate RNA clustering (Fig. 4f, left).In this picture, r(CAG) 20  Intra-condensate RNA clustering drives a liquid-to-solid phase transition What are the material properties of condensates containing percolated RNA clusters?Time-lapse microscopy suggests that the shell phase of the aged condensates containing TERRA clusters can undergo fusion, signifying liquid-like properties (Fig. 1d, Supplementary Video 1).However, these condensates are in contact with the glass surface, which can in uence their fusion kinetics substantially 72 .To examine the dynamical behavior of (TERRA) 10 containing RGG-d(T) 40 condensates quantitatively as a function of age, we employed controlled condensate fusion using optical tweezers 73,74 .We trapped two condensates using a dual-trap optical tweezer and initiated condensate fusion while recording the uorescence of FAM-(TERRA) 4 and Cy5-d(T) 40 simultaneously.At 20 minutes after the condensate preparation, when both TERRA and d(T) 40 are uniformly distributed throughout the droplets, they undergo fusion with a fusion relaxation time (τ) of 18.40 ± 0.02 ms/µm.The components were homogeneously mixed after the condensate coalescence was completed (Fig. 5a; Supplementary Video 5).However, for condensates aged for 150 minutes, distinct RNA clusters were visible within condensates (Fig. 5a; Supplementary Video 6).These aged condensates containing RNA clusters were still able to fuse indicating that the condensate shell is dynamic and behaves as a terminally viscous liquid.Force relaxation analysis showed that fusion of the outer shell of aged condensates occurs more rapidly with a fusion relaxation time of 3.87 ± 0.06 ms/µm (Fig. 5a).However, the irregular RNA clusters within the condensate remained demixed at all conditions, indicating that they are in a different material state than RGG-d(T) 40 rich phase (Fig. 5a).We tested this directly by designing a condensate dissolution assay, which would preferentially dissolve the non-percolated components (Supplementary Fig. 15a).We observed that addition of 0.5 µl of 5 M NaCl is su cient to rapidly dissolve condensates formed by RGGd(T) 40 (Supplementary Fig. 15b).However, NaCl addition to aged RGG-d(T) 40   15c; Supplementary Video 9).We also probed the translational mobility of each component in the condensate as a function of age by uorescence recovery after photobleaching (FRAP; Fig. 5c).We chose (TERRA) 4 as a representative RNA for these measurements which forms intra-condensate clusters by 8 hours (Fig. 3a, c).FRAP analysis reveals that at early time points before the onset of visible RNA clusters (< 4 hours), all three components are relatively dynamic (Fig. 5c; Supplementary Fig. 16; Supplementary Videos 10, 11, 12).However, FRAP traces of TERRA, but not RGG and d(T) 40 , show a progressive drop in recovery with condensate age (Fig. 5c; Supplementary Fig. 16; Supplementary Videos 13, 14, 15).
The reduced translational mobility of TERRA could result from the formation of dynamically arrested intra-condensate RNA networks, driving a liquid-to-solid phase transition.To directly probe the material properties of condensates, we employed video particle tracking (VPT) nanorheology using 200 nm uorescently labeled beads 8 (Fig. 5d).The mean squared displacement (MSD) pro les of the probe particles have distinct characteristics for Newtonian liquids, viscoelastic uids with terminal viscous behavior, and Kelvin-Voigt solids with terminal elastic behavior 12,75 (Fig. 5d, right).VPT measurements reveal that freshly prepared RGG-d(T) 40 condensates containing (TERRA) 10 display material properties similar to a Maxwell uid 9 with a terminal viscosity of 35.2 ± 4.85 Pa.s (Fig. 5e-h; Supplementary Video 16).Upon physical aging, the probe particles within condensates are caged as evidenced by a narrower spread of the particle trajectory (Fig. 5f).Correspondingly, the ensemble-averaged MSD pro les showed dramatic differences at times beyond the emergence of RNA clusters, e.g., t > 60 minutes (Fig. 5g).We nd that this is an emergent property of the (TERRA) 10 containing RGG-d(T) 40 condensate system since the two-component condensate system composed of RGG and d(T) 40 does not show time-dependent changes in MSD pro les (Supplementary Fig. 17).Further measurements of MSD pro les in RGG-d(T) 40   condensates containing (TERRA) 10 revealed a dramatic arrest of the probe particles at time points, t = 30 and 45 minutes, at which the microscale RNA clusters were not clearly visible (Fig. 5g; Supplementary Fig. 18; Supplementary Video 17).The dynamical slowdown of probe particles prior to the emergence of microscale RNA aggregates may indicate the formation of nanoscale pre-percolation clusters of TERRA 76 .Upon further aging, the ensemble-averaged MSDs show a plateauing behavior at longer lag times indicating a terminally elastic response reminiscent of Kelvin-Voigt solids 12 (Fig. 5g; observation time ≥ 150 minutes).This may stem from the caging of beads due to the onset of percolated RNA clusters (Fig. 5f).The estimated terminal viscosity of the condensates at 90 minutes is 489.3 ± 69.3 Pa.s (Fig. 5h), which is an order of magnitude higher than condensates at 15 minutes.At 150 minutes after sample preparation, the bead motions were completely arrested, which is a characteristic property of terminally solid material 12 (Supplementary Video 18).
We further performed nano-rheology measurements with (mut-TERRA) 10 (Fig. 2e, f), which lacks the ability to undergo intra-condensate percolation (Fig. 2g; Supplementary Fig. 4, 5).We observed that (observation time ~ 30 minutes) RGG-d(T) 40 condensates containing (mut-TERRA) 10 behave as a Maxwell uid with viscosity 21.5 ± 3.1 Pa.s, which is slightly lower than WT (TERRA) 10 (Fig. 5i, j; Supplementary Fig. 19).Importantly, no discernable changes in viscoelastic properties was observed for these condensates over the same period of time (4 hours; Fig. 5i, j; Supplementary Fig. 19) where the WT TERRA containing condensates undergo complete dynamical arrest.These results show that selectively inhibiting the percolation ability of the RNA through sequence perturbations abrogates RNA percolationdriven condensate dynamical arrest.

RNA-binding protein G3BP1 inhibits intra-condensate RNA clustering
In cells, RNP granules are comprised of diverse RNA and protein species with an assortment of sequence-speci c and non-speci c interactions 77,78,79,80 .Based on our results on age-dependent intracondensate RNA clustering, we reasoned that introducing additional components in these condensates that can compete with homotypic RNA-RNA interactions may inhibit RNA cluster formation.We tested this idea rst by employing an anti-sense oligonucleotide [ASO; sequence: r(CCCUAA)] targeting (TERRA) 10 .ASO-treated condensates did not show any signs of RNA clusters even after 24 hours in our uorescence microscopy experiments (Fig. 6a, b; Supplementary Fig. 20).VPT-based nanorheology con rmed the absence of nanoscale clusters (Fig. 6c; Supplementary Fig. 21) as no substantial change in condensate viscosity was observed during 24 hours of aging (Fig. 5f).These results suggest that selectively targeting homotypic RNA-RNA interactions can buffer against RNA clustering-mediated liquidto-solid phase transition.Could such buffering effects be also imparted by multivalent RBPs with broad speci city for a diverse repertoire of RNA?We tested this idea utilizing G3BP1, which is a core scaffolding RBP of cytoplasmic stress granules 81,82,83 .G3BP1 binds to RNAs through a folded RNA recognition motif and disordered Arg/Gly-rich domain (Fig. 6d).In the presence of 10 µM G3BP1, we observed that (TERRA) 10 containing RGG-d(T) 40 condensates do not show the emergence of RNA clusters after aging (within 24 hours).All components remain homogenous and the cluster sizes derived from SAC analysis show no substantial change in their spatial distribution (0.14 ± 0.01 µm at 15 minutes and 0.14 ± 0.01 µm at 24 hours; 1 pixel = 0.09765 µm) (Fig. 6e, f; Supplementary Fig. 23).
Earlier, our nano-rheology experiments on (TERRA) 10 containing RGG-d(T) 40 condensates (Fig. 5g, h) revealed signs of dynamical arrest and a substantial increase in viscosity even before microscale RNA clusters were visible.To quantify the effects of G3BP1 on the rheology of these condensates, we probed the time-dependent changes in condensate material properties using VPT.MSD pro les and viscosity measurements reveal that despite the absence of microscale RNA clusters, G3BP1 containing condensates undergo a progressive dynamical slowdown (Fig. 6g; Supplementary Fig. 24).There is a concomitant increase in viscosity from 42.3 ± 9.0 Pa.s for condensates at 30 minutes to 154 ± 22.3 Pa.s within 3 hours of condensate preparation (Supplementary Fig. 24).However, condensate aging is substantially slower in the presence of G3BP1 (Fig. 6h).Therefore, although microscale RNA clusters are disfavored in the presence of G3BP1, our rheology measurements suggest that there may be nanoscale RNA clustering that contributes to increased viscoelasticity of aged condensates.
Nonetheless, G3BP1 can increase the activation energy barrier of intra-condensate (TERRA) 10 clustering.
We further tested the generalizability of the observed buffering effect of G3BP1 with GC-rich repeat RNAs, r(CAG) 31 and r(CUG) 47 , that spontaneously form microscale clusters in RGG-d(T) 40 condensates (Fig. 4).In the presence of G3BP1, both of these RNAs formed homogeneous condensates (Fig. 6i-l; Supplementary Fig. 23).Furthermore, in the case of (CAG) 31 , no microscale clusters were observed even at 24 hours age where the cluster size = 0.39 ± 0.02 µm, close to the detection limit (1 pixel = 0.2196 µm) (Fig. 6i, j; Supplementary Fig. 23).In the case of r(CUG) 47 we observed the formation of a few RNA foci at 24 hours (Fig. 6k, l; Supplementary Fig. 23).However, the total fraction of RNA, determined by intensitybased analysis, in these clusters were only ~ 6.65 ± 2.31% (Supplementary Fig. 25), whereas the same RNA was present almost exclusively (~ 100%) in the intra-condensate clusters in the absence of G3BP1 (Fig. 4d).Together, these results signify that G3BP1 can frustrate 84 RNA-RNA homotypic interactions in these condensates thereby disfavoring RNA clustering and preserving intra-condensate solubility of repeats RNAs.

Conclusions
Recently, it has been shown that biomolecular condensates can act as sites for pathological protein aggregation 21,22,23,24,25,86 .We now demonstrate that irreversible clustering of repeat expanded RNA molecules, a process widely implicated in many neurological disorders 37,64,65,66,67,68,69 , can be nucleated in multi-component protein-nucleic acid condensates.The underlying mechanism is sequence-speci c percolation transitions of RNA chains driven by homotypic RNA-RNA interactions.RNA percolation engenders an age-dependent liquid-to-solid phase transition of condensates.
Multivalent co-factors, such as ASO and RBPs, that compete with homotypic RNA-RNA interactions can increase the activation energy barrier of RNA clustering, thereby acting as inhibitors of this process.Two important implications, as outlined in Fig. 7, stem from our experiments reported in this study.
Firstly, dynamic frustration of homotypic interactions between RNA chains can increase the range of metastability of biomolecular condensates that are poised to undergo RNA percolation-driven physical aging.In this model, a multi-component biomolecular system is frustrated if the probability of minimizing its global free energy through coordinated optimization of all possible interaction modalities of constituent macromolecules is kinetically sluggish due to overlapping inter-molecular interactions 84, 87, 88, 89, 90 .For RNA containing RGG-d(T) 40 condensates, the RNA [e.g., (TERRA) 10 or r(CAG) 31 or r(CUG) 47 ] is initially kept frustrated through heterotypic interactions with the primary condensate components, despite having a strong propensity to self-associate as evidenced by its hysteretic phase behavior and irreversible clustering 43 (Fig. 2).However, with time, RNA-RNA homotypic interactions dominate, leading to age-dependent RNA clusters that demix from the uid phase formed by RGG and d(T) 40 .The onset of RNA clustering is determined by the driving force for RNA percolation, which is modulated by repeat RNA length and/or sequence perturbations that weaken base pairing and stacking interactions.Reducing the homotypic RNA-RNA interactions, and hence, the percolation propensity shifts the balance towards heterotypic interactions between RNA and condensate components over RNA self-assembly.Consequently, the cross-play of interactions between all components as opposed to the dominance of a singular intermolecular, homotypic interaction node may be essential for enhancing the metastability of multi-component biomolecular condensates.
Second, in the cases of strongly percolating RNA molecules [e.g., (TERRA) 10 or r(CAG) 31 or r(CUG) 47 ], G3BP1 reduces the propensity of RNA clustering suggesting that the solubility of RNAs in condensates is enhanced.Based on these observations, we propose that RNA binding proteins in complex biomolecular condensates may employ heterotypic RNA-protein interactions 91 as a regulatory mechanism to prevent RNAs from aberrant homotypic self-assembly.This phenomenon, termed heterotypic buffering 91 , was previously proposed as a mechanism to enhance the solubility of aggregation-prone proteins, which possess a strong preference for homotypic interactions and amyloid ber formation linked to diseases.Our study extends this thermodynamic framework to rationalize the biological roles of RBPs in regulating intra-condensate RNA aggregation.During cellular stress, polysomes are disassembled and polyA-tailed mRNAs are sequestered in stress granules, thereby globally inhibiting translation 92,93 .Intra-stress granule RNA percolation can compromise the disassembly of stress granules upon removal of stress, leading to the accumulation of irreversible granules that are cytotoxic 22,94 .Our results indicate that multivalent RNA binding proteins can effectively provide the rst line of defense against irreversible RNA clustering before ATP-dependent RNA helicases can actively engage in remodeling these assemblies 78 .
In summary, we report that percolation-driven irreversible RNA clustering can be enhanced in biomolecular condensates, leading to their liquid-to-solid phase transitions.This can be buffered by multivalent RBPs supporting liquid-phase condensate homeostasis.The insights gained from our study provide a complementary perspective on the role of RBPs in regulating aberrant RNA self-assembly in living cells.Pairwise line pro le analyses of (TERRA) 4 images with respect to RGG (e) and d(T) 40 (f) as a function of time (for corresponding images, see Supplementary Fig. 9).Each line pro le shown here is normalized with respect to the maximum intensity value, wherein all values were rst offset by the minimum intensity value.The composition of the ternary RGG-d(T) 40 condensate system is 1 mg/ml RNA, 5 mg/ml RGG, and 1.5 mg/ml d(T) 40 in a buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT.

Declarations Figures
In experiments utilizing uorescently labeled components, the concentration range is 250 nM to 500 nM.Each experiment was independently repeated at least three times.minutes after sample preparation but show some RNA clusters at an age of 24 hours.(l) The corresponding cluster size analysis is reported.The concentrations of the ASO and G3BP1 are 1 mg/ml and 10 μM, respectively.The composition of the condensate system used for imaging is 1 mg/ml RNA [0.45 mg/ml in the case of r(CUG) 47 ], 5 mg/ml RGG, and 1.5 mg/ml d(T) 40 .For the nanorheology measurements, the relative proportion of the condensate components was kept the same, but the overall concentration of each component was doubled to achieve a higher volume fraction of the dense phase.
Buffer composition for all experiments is 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT.In experiments utilizing uorescently labeled components, the concentration range is 250 nM to 500 nM.
Each experiment was independently repeated at least three times, except for the sample with G3BP1 in (h) that aged for 90 minutes, which was independently repeated two times.
condensates containing TERRA clusters showed only partial dissolution where the RGG-d(T) 40 rich shell phase dissolved immediately but the RNA clusters persisted (Fig. 5b; Supplementary Videos 7, 8).Notably, freshly prepared (TERRA) 10 containing RGG-d(T) 40 condensates lacking RNA clusters (15 minutes post preparation) were observed to dissolve completely by NaCl treatment (Supplementary Fig.

Figure 7 schematic
Figure 7 (a) A model of multi-component condensates formed by two RNAs with strong (as shown in green) and weak (as shown in red) percolation propensity, respectively, and an RBP.(b) Three possible scenarios of RNA percolation-driven condensate aging or a lack thereof in the presence of a multivalent RBP.(c) Zoomed-in views of the panels shown above.